Expression of full-length and splice forms of FoxP3 in rheumatoid arthritis.

OBJECTIVE: The aim of our study was to compare the presence of full-length and alternative splice forms of FoxP3 mRNA in CD4 cells from rheumatoid arthritis (RA) patients and healthy controls. METHODS: A quantitative real-time polymerase chain reaction (QRT-PCR) method was used to measure the amount of FoxP3 mRNA full-length and splice forms. CD4-positive T cells were isolated from peripheral blood from 50 RA patients by immunomagnetic separation, and the FoxP3 mRNA expression was compared with the results from 10 healthy controls. RESULTS: We observed an increased expression of full-length FoxP3 mRNA in RA patients when compared to healthy controls, as well as an increase in CD25 mRNA expression, but no corresponding increase in CTLA-4 mRNA expression. The presence of an alternative splice form of FoxP3 lacking exon 2 was confirmed in both RA patients and healthy controls, but with no significant difference in expression between the two groups. There was a positive correlation between the amount of FoxP3 mRNA and the clinical inflammation parameters C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and a negative correlation between FoxP3 mRNA and the dose of methotrexate (MTX) given to the patients. CONCLUSION: RA patients express more full-length FoxP3 than healthy controls in peripheral blood CD4-positive cells, suggesting an increased number of regulatory T cells (Tregs). However, no concomitant increase in CTLA-4 expression was seen. We therefore propose that the Tregs are left unable to suppress the ongoing inflammation due to a deficiency in CTLA-4 needed for cell contact-dependent suppression.

Tissue viscoelasticity is related to tissue composition but may not fully predict the apparent-level viscoelasticity in human trabecular bone - An experimental and finite element study.

Trabecular bone is viscoelastic under dynamic loading. However, it is unclear how tissue viscoelasticity controls viscoelasticity at the apparent-level. In this study, viscoelasticity of cylindrical human trabecular bone samples (n=11, male, age 18-78 years) from 11 proximal femurs were characterized using dynamic and stress-relaxation testing at the apparent-level and with creep nanoindentation at the tissue-level. In addition, bone tissue elasticity was determined using scanning acoustic microscope (SAM). Tissue composition and collagen crosslinks were assessed using Raman micro-spectroscopy and high performance liquid chromatography (HPLC), respectively. Values of material parameters were obtained from finite element (FE) models by optimizing tissue-level creep and apparent-level stress-relaxation to experimental nanoindentation and unconfined compression testing values, respectively, utilizing the second order Prony series to depict viscoelasticity. FE simulations showed that tissue-level equilibrium elastic modulus (Eeq) increased with increasing crystallinity (r=0.730, p=.011) while at the apparent-level it increased with increasing hydroxylysyl pyridinoline content (r=0.718, p=.019). In addition, the normalized shear modulus g1 (r=-0.780, p=.005) decreased with increasing collagen ratio (amide III/CH2) at the tissue-level, but increased (r=0.696, p=.025) with increasing collagen ratio at the apparent-level. No significant relations were found between the measured or simulated viscoelastic parameters at the tissue- and apparent-levels nor were the parameters related to tissue elasticity determined with SAM. However, only Eeq, g2 and relaxation time τ1 from simulated viscoelastic values were statistically different between tissue- and apparent-levels (p<.01). These findings indicate that bone tissue viscoelasticity is affected by tissue composition but may not fully predict the macroscale viscoelasticity in human trabecular bone.

Collagen structure in skin from fibromyalgia patients.

The distribution and amount of collagen in skin from a non-tender-point area from fibromyalgia patients was assessed by quantitative analysis of amino acids and by electron and light microscopy. Skin biopsies were obtained from the front of the thigh of 27 females who fulfilled the American College of Rheumatology criteria of fibromyalgia and from eight control subjects who were matched for gender, age and physical activity. Amino acids were determined by high-performance liquid chromatography. Electron and light microscopic investigations were carried out to examine tissue structure. Among the collagen-related amino acids, the mean number of hydroxyproline residues per 1,000 residues was 52.5 and 63.4 in fibromyalgia patients and control subjects, respectively (p = 0.050); proline residues were 81.7 and 110.0 (p = 0.006); and hydroxylysine residues were 14.7 and 10.1 (p = 0.002). The total amount of skin protein in proportion to dry tissue weight was 83.4% and 72.6% in fibromyalgia and controls, respectively (p = 0.037). The overall microscopic picture was normal. The lamellar structure of the perineurium and a deficiency in collagen packing in the endoneurium was observed more frequently and to a larger extent in fibromyalgia patients than in controls. In conclusion, there are some differences between the amino acid composition of skin proteins in fibromyalgia patients compared with controls. The amount of collagen may be lower in skin from fibromyalgia patients, and collagen packing in the endoneurium may be less dense.

Biochemical and ultrastructural changes in rabbit sclera after treatment with 7-methylxanthine, theobromine, acetazolamide, or L-ornithine.

AIMS: To examine a possible effect of 7-methylxanthine, theobromine, acetazolamide, or L-ornithine on the ultrastructure and biochemical composition of rabbit sclera. METHODS: Groups of pigmented rabbits, six in each group, were dosed during 10 weeks with one of the substances under investigation, and one untreated group was the control. Samples of anterior and posterior sclera were taken for determination of hydroxyproline, hydroxylysine, proline, proteoglycans, uronic acids and dermatan sulphate, chondroitin sulphate, and hyaluronic acid. Sections were examined with electron microscopy, and the diameter of the individual collagen fibrils was measured. RESULTS: Treatment with theobromine produced a significant increase in the contents of hydroxylysine, hydroxyproline, and proline in both anterior and posterior sclera, while 7-methylxanthine increased the contents of hydroxyproline and proline selectively in posterior sclera. Acetazolamide, on the other hand, significantly decreased the contents of hydroxyproline and proline in samples from anterior sclera. Uronic acids in both anterior and posterior sclera were significantly reduced by treatment with 7-methylxanthine, and L-ornithine significantly reduced uronic acids in posterior sclera. An inverse correlation between contents of hydroxyproline and uronic acids was found. The mean diameter of collagen fibrils was significantly higher in the posterior sclera from rabbits treated with 7-methylxanthine or theobromine, and significantly lower in rabbits treated with acetazolamide or L-ornithine compared with controls. In the anterior sclera, fibril diameter was significantly reduced in all treatment groups compared with controls. A positive, significant correlation between fibril diameter and content of hydroxyproline and proline was found in posterior sclera. CONCLUSION: 7-Methylxanthine, a metabolite of caffeine, increases collagen concentration and the diameter of collagen fibrils in the posterior sclera, and may be useful for treatment or prevention of conditions associated with low level and/or inferior quality of scleral collagen, such as axial myopia, chronic open angle glaucoma, and possibly neovascular age related macular degeneration. The apparent loss of collagen induced by chronic treatment with acetazolamide should be taken into consideration as a potentially harmful side effect. These results may indicate that scleral biochemistry and ultrastructure are influenced by the retinal pigment epithelium. One possible explanation is that the scleral fibroblasts which produce the collagen are sensitive to changes in the physiological electric field created by the retinal pigment epithelium.

Evaluation of chronic venous disease in the lower limbs: comparison of five diagnostic methods.

To compare the usefulness of five diagnostic methods in ensuring deep vein patency, and in demonstrating site(s) of incompetence, 39 patients with clinical signs of chronic venous disease of a leg were included in a study of deep, superficial and perforator veins using triplex ultrasound (TUS), ascending phlebography (AP), descending phlebography (DP), continuous wave Doppler (CWD) and ambulatory strain gauge plethysmography (ASGP). One patient withdrew from the study. It was not possible to use all five methods in all 38 cases, and the methods could only be used partly in some cases. TUS, which allows anatomical, morphological and functional evaluation of the venous system, was chosen as the reference method. There was poor agreement between TUS and AP, and no agreement between TUS and ASGP, in the diagnosis of venous occlusion. AP demonstrated reflux (abnormal valves) in 7 of 22 patients with competent veins at TUS, and missed reflux in 13 of 15 patients with incompetent veins. Similarly, CWD overdiagnosed reflux in 13 of 20 patients and missed the reflux in 3 of 14 patients. DP was only technically possible in 11 patients. ASGP diagnosed venous reflux in all patients with incompetent deep veins, but also indicated deep vein or perforator vein reflux in all but one patient with competent deep veins. The agreement between TUS and the other methods in evaluating reflux in the deep veins was not better than that expected to occur by chance, Cohen's kappa being less that 0.20. It is concluded that AP, CWD and ASGP are of little value in the work-up of patients with deep venous insufficiency.

Urinary markers of altered collagen metabolism in fibromyalgia patients.

OBJECTIVE: To assess the metabolism of collagen in fibromyalgia (FM) patients, and to compare the occurrence of collagen metabolism markers to the severity of FM symptoms. METHODS: Morning urine was collected from 27 FM women fulfilling the American College of Rheumatology (ACR) criteria for FM, and from seven controls. FM patients completed the Fibromyalgia Impact Questionnaire (FIQ). Bone mineral density (BMD), isokinetic muscle strength in knee and elbow, and hand-grip strength were measured. Urinary concentrations of collagen type I cross-linked C-telopeptide (CTX-I) and collagen type II cross-linked C-telopeptide (CTX-II) were determined by enzyme-linked immunosorbent assay (ELISA). Pyridinoline (Pyd) and deoxypyridinoline (Dpd) were determined by liquid chromatography, and hydroxyproline (Hyp) by spectrophotometry. All concentration data were normalized to creatinine. RESULTS: Mean values in the FM group and the control group, respectively, were: urinary CTX-I 246.8 and 337.5 microg/mmol (p = 0.060); CTX-II 110.4 and 185.1 ng/mmol (p = 0.035); Pyd 56.1 and 52.3 nmol/mmol (NS); Dpd 15.1 and 14.0 nmol/mmol (NS); Pyd : Dpd ratio 4.05 and 3.96 (NS); Hyp 26.1 and 21.1 micromol/mmol (NS). Significant inverse correlations were seen between CTX-I and the intensity of fatigue, and between CTX-II and anxiety. An inverse correlation between CTX-I and muscle strength was apparent, but relied on extreme values from one patient, and no significant correlation was found between CTX-I or CTX-II and tender points or BMD in the FM group. CONCLUSIONS: Low urinary concentrations of CTX-II and CTX-I and normal levels of Pyd and Dpd were found in FM, but their relationship to the intensity of FM symptoms was unclear.

Collagen and muscle pathology in fibromyalgia patients.

OBJECTIVE: To measure collagen concentration and search for muscle pathology in muscle non-tender-point areas from fibromyalgia (FM) patients. METHODS: Muscle biopsies were obtained from m. vastus lateralis of 27 carefully selected, female fibromyalgia patients, and from eight age-matched female control subjects. Amino acids were determined by HPLC and electron microscopy was performed. RESULTS: The FM patients had lower hydroxyproline and lower total concentration of the major amino acids of collagen than the controls. No significant difference was seen in the concentration of the major amino acids of myosin or of total protein. Electron microscopy showed no significant differences between FM patients and controls although atrophied muscle fibrils occurred in FM patients only, but frequencies were not significantly different. CONCLUSION: Fibromyalgia patients had a significantly lower amount of intramuscular collagen. This may lower the threshold for muscle micro-injury and thereby result in non-specific signs of muscle pathology.